Biofilm disassembly and dispersal play a key role in disease spread, but studying these processes with microscopy and related imaging techniques is challenging. The fluorescent proteins traditionally used to label cells lose functionality in the oxygen-deprived biofilm environment, inhibiting close observation of the biofilm dispersal process.

To resolve this issue, a team at Carnegie Mellon University labeled cells with oxygen-independent fluorogen-activating proteins (FAPs) and cognate far-red dyes. The FAPs and far-red probes remain functional throughout biofilm development and dispersal, allowing bacterial biofilms to be visualized in real time, over the long term, at a resolution higher than what was previously possible.

“No one had been able to image biofilm dispersal with the sort of resolution that we were able to achieve,” professor Drew Bridges said. “And it is because of FAP labeling technology.”

The scarcity of oxygen in biofilms prevents traditional fluorescent proteins from emitting light. FAPs, which were developed at Carnegie Mellon in 2008 and emit fluorescent light only when bound to a fluorogen, may provide an alternative to oxygen-dependent fluorescent labeling.

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A series of microscopic images shows dispersion of vibrio cholerae bacteria over time. Courtesy of Carnegie Mellon University.

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“When I got to Carnegie Mellon, I learned about FAPs,” Bridges said. “And they’re the perfect alternative because their mechanism is very different from how other fluorescent proteins work. They’re not sensitive to oxygen limitation.”

Working with project scientist Robert van de Weerd, the Bridges lab incorporated FAPs into the genome of the Vibrio cholerae bacteria. The researchers added malachite green-derived fluorogens to the bacterial colony. The fluorogens bound to the FAPs and emitted far-red fluorescence. The far-red region of the visible spectrum is typically less toxic to living organisms and safer for imaging tissues.

Using spinning-disc confocal microscopy, the team followed cells in the V. cholerae biofilms as the cells disassembled and dispersed. Intense, stable, far-red fluorescence from the cells enabled the researchers to examine large biofilm communities at single-cell resolution throughout the dispersal process.

The team found that cells begin their dispersal from the biofilm periphery. It also found that biofilm dispersal is heterogenous, with about 20-25% of the cells remaining in the biofilm once the dispersal phase is complete. Further investigation will help determine whether these cells remain behind because they are trapped or for a different reason.

Far-red FAP imaging also revealed the development of dispersal “hot spots” — localized, dynamic regions where cells exhibited large outward displacements compared to cells in the surrounding regions. The researchers also observed that some cells in the biofilm periphery did not disperse and instead compressed toward the biofilm core. They hypothesize that cells could be a major mechanical component in the biofilm, and, as the cells start to disperse, the overall structure collapses.

The results suggest that heterogeneous cell dispersal from biofilms could be common in bacteria. The team plans to test this hypothesis by applying the FAP labeling technology to other notorious biofilm formers.

The FAP-fluorogen labeling approach could lead to a better understanding of how pathogens spread between infection sites. It could be useful in high-resolution studies of microbial dynamics in diverse areas of microbiology. The oxygen independence of FAP fluorescence combined with the fluorogen’s far-red spectral properties could, for example, enable the detailed interrogation of microbiome biogeography in live animals.

Different FAP-fluorogen pairs have been developed to span the visible spectrum. These probes could, for example, be used to tag proteins of interest, label multiple organisms simultaneously, or monitor gene expression in complex microbial communities.

The research was published in PLOS Biology (www.doi.org/10.1371/journal.pbio.3002928).

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